The objective of the proposed research is to define the role of T lymphocytes in the regulation of the humoral immune response. We have reported the existence of three factors which can act nonspecifically to amplify the B cell antibody response in vitro. These enhancing factors (EF) will be generated in tissue culture (a) by mixing histoincompatible murine thymocytes, or purified T lymphocytes, (b) by specific antigenic stimulation between human lymphocyte cell lines. Another soluble EF will be recovered from the plasma of mice 4 days after infection with Friend leukemia virus. T cells and B cells will be purified from the spleens of antigen-primed mice, and normal mice, by passage of cell suspensions through nylon wool columns. Additionally, using an adoptive transfer system, mice will be x-irradiated, reconstituted with thymus cells and antigen, and their spleens used as a source of "educated" T cells. Similarly, B cells will be recovered from the spleens of adult thymectomized, irradiated, bone marrow-protected mice. The murine and human EFs will be purified by a combination of techniques including Amicon membrane separation, ammonium sulfate fractionation, Sepahdex gel filtration, ion exchange and affinity chromatography, and polyacry lamide gel electrophoresis. The activity of the purified factors will be assayed by addition of EF to cultures of T cell- depleted, B lymphocytes stimulated with heterologous erythrocytes of other antigens. The IgM and IgG PFC responses of such cultures will be compared in the presence and absence of EF. EF activity in vivo will be similarly studied by following antibody titer, splenic PFC formation and colony-forming unit proliferation in antigen-stimulated mice. Anti-EF antibody will be used to specifically inhibit these immune responses. To study the mechanism of action of the mediators, we will examine DNA synthesis, antigen-binding, I125-EF binding, and distribution of Ig receptors in antigen or mitogen stimulated B cells and T cells. The role of macrophages in the enhancement phenomenon will also be determined. Furthermore, the presence and activity of an inhibitory substance liberated from suppressor T cells, which antagonizes the effects of EF, will be characterized. These studies will increase our knowledge of the cellular interactions and biochemical mechanisms which govern the regulation of antibody synthesis.